Hepatitis C virus (HCV) results in persistent infection in up to 80% of infected individuals and causes a wide spectrum of liver diseases. Liver disease related to chronic HCV infection is now the leading indication for liver transplantation in the United States. Currently there is no preventive vaccine and available antiviral therapy has only limited clinical success. The understanding of molecular mechanisms during the life cycle of HCV is hampered by the lack of a reliable cell culture system. While recent success in HCV cell culture has been achieved using an "infectious clone" approach with a HCV genotype 2a strain (JFH1), this system has not been replicated with HCV strains other than JFH1, a strain which was derived from a patient with fulminant hepatitis due to HCV infection, a very rare clinical event. Analysis of HCV isolates from this individual displayed an extremely homologous viral population in which the JFH1 strain, assembled from multiple overlapped PCR fragments, reaches the maximum similarity to the authentic viral sequence. Otherwise full-length HCV sequences obtained in the same way represent population sequences, defined as the sequences derived from PCR product directly. We have recently provided evidence that population sequences may be artificial due to quasispecies nature of RNA viruses , We therefore hypothesize that HCV cell culture systems could be established and/or improved for HCV genotypes other than JFH1 through the direct use of authentic viral sequences. This hypothesis will be tested with isolates of HCV genotype 1a in the following specific aims: Specific Aim 1: Generation of authentic full-length HCV sequences from clinical samples. The clinical serum samples used to generate authentic full-length HCV sequences were selected from our serum bank under the guidance of phylogenetic analysis. From our serum bank, we have completed genetic studies for 130 patients infected with HCV genotype 1, which have formed three subgroups within HCV genotype 1a branch in the phylogenetic tree. We selected one serum sample from each HCV 1a subgroup, which showed minimal genetic diversity but high viral RNA levels. Additionally, a serum sample for HCV Hutchinson strain (H77) was kindly provided by Dr. Robert Purcell in NIH. These four serum samples will be used to generate full-length HCV sequences at the quasispecies level by using a novel long RT-PCR (LRP) and cloning technology. Specific Aim 2: Screening and establishing HCV cell culture with authentic full-length HCV sequences. The full-length HCV sequences generated in specific aim 1 will be used to establish HCV cell culture through an "infectious clone" approach, as was done with the JFH1 strain. Phylogenetic analysis will be performed to select appropriate clones for HCV cell culture. The HCV specific infectivity will be quantitatively estimated for these selected viral sequences as well as existing HCV infectious clones, including JFH1 and H77 strains. The potential genomic and structural determinants will be explored through a comparative analysis among these clones, including existing infectious clones derived from H77. The "infectious clone" approach has become a routine method to study RNA viruses and has been successful for many flaviviruses and pestiviruses. To some extent, it is not readily understood why it has been so difficult to make infectious clones of HCV. The availability of novel LRP and cloning technology will allow us to address this question in lieu of viral genetic heterogeneity.